Bio-Rad Bio-Plex Pro™ Human Chemokine Assays - Manual (Page 18)

16
Lysates
The Bio-Plex cell lysis kit is required for lysate preparation (available
separately, catalog #171-304011 and #171-304012). Refer to bulletin #5297
for a list of published articles on cytokine analysis in tissue samples.
1. Prepare the cell or tissue lysates according to the instructions
provided with the Bio-Plex cell lysis kit. The protease inhibitors
factor I and factor II are included in the kit. PMSF needs to be added
to lysis buffer at a final concentration of 2 mM. The lysates should be
free of particulate matter.
2. Determine the total protein concentration of the lysate. It may be
necessary to test lyse your samples with different volumes of lysing
solution to obtain the specified protein concentration range.
3. Dilute at least 1:2 in sample diluent + 0.5% BSA, to a final protein
concentration of 200–900 μg/ml. For analytes with high expression, a
lysate protein concentration as low as 50 μg/ml may be sufficient.
Note: For optimum antibody binding during sample incubation, it is
important to dilute lysates as much as possible to reduce the detergent
concentration.
4. If the lysate is not tested immediately, store at –20°C to –70°C. Avoid
repeated freeze-thaw cycles.
7. Prepare Coupled Beads
1. Use Tables 6–7 or the Calculation Worksheet on page 36 to calculate
the volume of coupled beads and assay buffer needed to prepare a
1x stock.
2. Add the required volume of Bio-Plex ® assay buffer to a 15 ml
polypropylene tube.